Chloride Is essential for capacitation and for the capacitation-associated increase in tyrosine phosphorylation.
After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of the oviductal fluid. It is well established that capacitation requires Na(+), HCO(3)(-), Ca(2+), and a cholesterol acceptor; however, little is known about the function of Cl(-) during this important process. To determine whether Cl(-), in addition to maintaining osmolarity, actively participates in signaling pathways that regulate capacitation, Cl(-) was replaced by either methanesulfonate or gluconate two nonpermeable anions. The absence of Cl(-) did not affect sperm viability, but capacitation-associated processes such as the increase in tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, the zona pellucidae-induced acrosome reaction, and most importantly, fertilization were abolished or significantly reduced. Interestingly, the addition of cyclic AMP agonists to sperm incubated in Cl(-)-free medium rescued the increase in tyrosine phosphorylation and hyperactivation suggesting that Cl(-) acts upstream of the cAMP/protein kinase A signaling pathway. To investigate Cl(-) transport, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na(+)/K(+)/Cl(-) cotransporters (NKCC), inhibited all capacitation-associated events, suggesting that these transporters may mediate Cl(-) movements in sperm. Consistent with these results, Western blots using anti-NKCC1 antibodies showed the presence of this cotransporter in mature sperm.
Wertheimer
Eva V
EV
Department of Veterinary and Animal Science, Paige Laboratories, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Salicioni
Ana M
AM
Liu
Weimin
W
Trevino
Claudia L
CL
Chavez
Julio
J
Hernández-González
Enrique O
EO
Darszon
Alberto
A
Visconti
Pablo E
PE
D020101:Acrosome Reaction / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005260:Female; D005665:Furosemide / Q000494:pharmacology; D008297:Male; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D064506:Solute Carrier Family 12, Member 2; D013075:Sperm Capacitation / Q000187:drug effects / Q000502:physiology*; D013081:Sperm Motility / Q000187:drug effects / Q000502:physiology*; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D015044:Zona Pellucida / Q000378:metabolism
D002712:Chlorides; C578878:Slc12a2 protein, mouse; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D002034:Bumetanide; D014443:Tyrosine; D005665:Furosemide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases